The overall objectives of the grant which this application proposes to supplement are to use 18O exchange techniques to develop a method for measuring the permeability of membranes to CO2 and HCO3 as well as measuring intracellular carbonic anhydrase activities. A related objective is to elucidate the mechanism of catalysis by carbonic anhydrase. The specific goals of the research proposed in this application include the construction and use of rapid flow techniques to overcome the problem of measuring fast 18O depletion from CO2 in the presence of red cells. Such an apparatus will allow accurate measurement of exchange rates which are too rapid for us to measure with current techniques. The resulting information will be utilized in the appropriate rate equations to obtain values for the permeability constants of the human red cell membrane to CO2 and HCO3 and the intracellular activity of carbonic anhydrase. It has been determined that buffers in solution participate as proton transfer agents in the catalytic pathway for CO2 hydration by carbonic anhydrase. We propose to investigate the possibility that His 63 in the active site crevice of human carbonic anhydrase C participates as a proton carrier, shuttling protons between buffers in solution and the active site. This will be approached by carboxyketoethylating His 63, a known procedure accomplished with bromopyruvate, and measuring 18O exchange catalyzed by the modified enzyme. By measuring the extent to which 18O is incorporated in 13C-labeled CO2 species in the catalysis, we aim to estimate the residence time in the active site of this essential oxygen which is incorporated into bicarbonate during the hydration reaction. We will also be able to measure the pH dependence of this residence time.